Protein Stabilized Antimicrobial Composition Formed by Melt Processing

ABSTRACT

A method for forming an antimicrobial composition that includes mixing an antimicrobially active botanical oil (e.g., thymol, carvacrol, etc.) and protein within a melt blending device (e.g., extruder) is provided. Despite the problems normally associated with melt processing proteins, the present inventors have discovered that the processing conditions and components may be selectively controlled to allow for the formation of a stable, melt-processed composition that is able to exhibit good mechanical properties. For example, the extrusion temperature(s) and shear rate employed during melt blending are relatively low to help limit polypeptide dissociation, thereby minimizing the impact of aggregation and embrittlement. While the use of such low temperature/shear conditions often tend to reduce mixing efficiency, the present inventors have discovered that a carrier fluid may be employed to enhance the ability of the botanical oil to flow into the internal structure of the protein where it can be retained in a stable manner. The composition is also typically anhydrous and generally free of solvents. In this manner, the protein will not generally disperse before use and prematurely release the botanical oil.

BACKGROUND OF THE INVENTION

Certain types of botanical oils, such as thymol and carvacrol, are knownto be environmentally friendly and effective in combatingmicroorganisms. Unfortunately, however, the use of such oils has beenlimited in many commercial applications (e.g., wipes) due to their highvolatility and instability in the presence of oxygen. Attempts toovercome this problem often involve the use of a larger amount of thebotanical oils to prolong antimicrobial activity. Regrettably, this justleads to another problem in that high concentrations of essential oilscan cause damage to certain types of food products, such as fruit. Otherattempts have involved the encapsulation of the oil component withcertain types of polymers, such as proteins. For example, an articleentitled “Encapsulation of Essential Oils in Zein NanosphericalParticles” (Parris, et al., J. Agric. Food Chem. 2005, 53, 4788-4792)broadly describes the encapsulation of thymol in zein nanospheres bymixing the oil with zein particles in the presence of a solvent (e.g.,ethanol). The particles are said to be useful for oral or injectableadministration of biological materials into the body. Another articleentitled “Controlled Release of Thymol from Zein Based Film”(Mastromatteo, et al., J. innovative Food and Emerging Technologies2009, 10, 222-227) broadly describes films formed by dissolving cornzein and glycerol into ethanol, and thereafter adding thymol to form asolution. The solution is poured into a Petri dish and dried to form thefilm.

One problem with the techniques described above is that they generallyrely on solvents (e.g., ethanol) to help dissolve the botanical oil intoa solution. A disadvantage of the use of solvents is that both thebotanical oil and protein must be soluble in a common solvent system,which puts a limit on what type of components may be employed in thecomposition. Also, solvent-based solutions require a substantial amountof time, energy, and material for processing. Still further, a portionof the botanical oil may escape from the solution when the solvent isevaporated, which requires the use of a greater amount of the oil thanwould normally be needed. Notwithstanding the above, the ability to usea “solventless” process is complicated by the tendency of proteins tolose their flow properties when exposed to the intense shear andelevated temperature normally associated with melt processing. Forexample, proteins may undergo a conformational change (“denaturation”)that causes disulfide bonds in the polypeptide to dissociate intosulfhydryl groups or thiyl radicals. Sulfhydryl groups form whendisulfide bonds are chemically reduced while mechanical scission ofdisulfide bonds causes thiyl radicals to form. Once dissociated,however, free sulfhydryl groups randomly re-associate with othersulfhydryl groups to form new disulfide bond between polypeptides. Thiylradicals can also randomly re-associate with other thiyl radicalsforming new disulfide bonds or thiyl radicals can react with other aminoacid functionality creating new forms of cross-linking betweenpolypeptides. Because one polypeptide contains multiple thiol groups,random cross-linking between polypeptide leads to formation of an“aggregated” polypeptide network, which is relatively brittle and leadsto a loss of flow properties.

As such, a need currently exists for a solventless process for forming astable composition that contains an antimicrobially active botanicaloil.

SUMMARY OF THE INVENTION

In accordance with one embodiment of the present invention, a method forforming an antimicrobial composition is disclosed that comprisesdispersively blending a botanical oil, protein, and carrier fluid withina melt blending device at a temperature of from about 20° C. to about100° C. and a shear rate of from about 1 to about 100 Pascal-seconds.Botanical oils constitute from about 0.1 wt. % to about 30 wt. % of thecomposition, proteins constitute from about 30 wt. % to about 95 wt. %of the composition, and carrier fluids constitute from about 1 wt. % toabout 50 wt. % of the composition.

In accordance with another embodiment of the present invention, amelt-processed antimicrobial composition is disclosed that comprises atleast one monoterpene phenol in an amount of from about 0.1 wt. % toabout 30 wt. %, at least one melt-processible plant protein in an amountof from about 30 wt. % to about 95 wt. %, and at least one carrier fluidin amount of from about 1 wt. % to about 50 wt. %. In yet anotherembodiment, a method for removing bacteria from a surface is disclosedthat comprises contacting the surface with a wipe that comprises afibrous material that contains the melt-processed antimicrobialcomposition.

Other features and aspects of the present invention are discussed ingreater detail below.

BRIEF DESCRIPTION OF THE FIGURES

A full and enabling disclosure of the present invention, including thebest mode thereof, directed to one of ordinary skill in the art, is setforth more particularly in the remainder of the specification, whichmakes reference to the appended figures in which:

FIG. 1 is an SEM microphotograph of a control sample containing 70 wt. %gluten and 30 wt. % glycerol;

FIG. 2 is an SEM microphotograph of a control sample containing 70 wt. %gluten and 30 wt. % glycerol;

FIG. 3 is an SEM microphotograph of Sample 3 of Example 1;

FIG. 4 is an SEM microphotograph of Sample 3 of Example 1; and

FIG. 5 is a graph of the results obtained in Example 3, in which the %thymol retained in water is shown for a period of time ranging from 0 to60 minutes.

Repeat use of references characters in the present specification anddrawings is intended to represent same or analogous features or elementsof the invention.

DETAILED DESCRIPTION OF REPRESENTATIVE EMBODIMENTS

Reference now will be made in detail to various embodiments of theinvention, one or more examples of which are set forth below. Eachexample is provided by way of explanation of the invention, notlimitation of the invention. In fact, it will be apparent to thoseskilled in the art that various modifications and variations may be madein the present invention without departing from the scope or spirit ofthe invention. For instance, features illustrated or described as partof one embodiment, may be used on another embodiment to yield a stillfurther embodiment. Thus, it is intended that the present inventioncovers such modifications and variations as come within the scope of theappended claims and their equivalents.

Generally speaking, the present invention is directed to a method forforming an antimicrobial composition that includes mixing anantimicrobially active botanical oil (e.g., thymol, carvacrol, etc.) andprotein within a melt blending device (e.g., extruder). Despite theproblems normally associated with melt processing proteins, the presentinventors have discovered that the processing conditions and componentsmay be selectively controlled to allow for the formation of a stable,melt-processed composition that is able to exhibit good mechanicalproperties. For example, the extrusion temperature(s) and shear rateemployed during melt blending are relatively low to help limitpolypeptide dissociation, thereby minimizing the impact of aggregationand embrittlement. While the use of such low temperature/shearconditions often tend to reduce mixing efficiency, the present inventorshave discovered that a carrier fluid may be employed to enhance theability of the botanical oil to flow into the internal structure of theprotein where it can be retained in a stable manner. The composition isalso typically anhydrous and generally free of solvents. In this manner,the protein will not generally disperse before use and prematurelyrelease the botanical oil.

Various embodiments of the present invention will now be described inmore detail below.

I. Components

A. Botanical Oil

Botanical oils are employed in the composition of the present inventionas antimicrobial actives. The oil may be an “essential” oil that isextracted from a plant. Likewise, the botanical oil may also be isolatedor purified from an essential oil, or it may simply be madesynthetically to mimic a compound derived from a plant (e.g.,synthetically made thymol). The botanical oils are generally soluble inlipids and believed to exhibit antimicrobial efficacy due to theirability to cause damage to the lipid component of the cell membrane inmicroorganisms, thereby inhibiting their proliferation. Essential oilsare derived from herbs, flowers, trees, and other plants, and aretypically present as tiny droplets between the cells of the plants andmay be extracted by methods known to those of skill in the art (e.g.,steam distillation, enfleurage (i.e., extraction using fat(s)),maceration, solvent extraction, or mechanical pressing). Examples ofsuitable essential oils for use in the present invention may include,for instance, anise oil, lemon oil, orange oil, oregano, rosemary oil,wintergreen oil, thyme on, lavender oil, clove oil, hops, tea tree oil,citronella oil, wheat oil, barley oil, lemongrass oil, cedar leaf oil,cedar wood oil, cinnamon oil, fleagrass oil, geranium oil, sandalwoodoil, violet oil, cranberry oil, eucalyptus oil, vervain oil, peppermintoil, gum benzoin, basil oil, fennel oil, fir oil, balsam oil, menthol,ocmea origanum oil, Hydastis carradensis oil, Berberidaceae daceae oil,Ratanhiae and Curcuma longa oil, sesame oil, macadamia nut oil, eveningprimrose oil, Spanish sage oil, Spanish rosemary oil, coriander oil,thyme oil, pimento berries oil, rose oil, bergamot oil, rosewood oil,chamomile oil, sage oil, clary sage oil, cypress oil, sea fennel oil,frankincense oil, ginger oil, grapefruit oil, jasmine oil, juniper oil,lime oil, mandarin oil, marjoram oil, myrrh oil, neroli oil, patchoulioil, pepper oil, black pepper oil, petitgrain oil, pine oil, rose ottooil, spearmint oil, spikenard oil, vetiver oil, or ylang ylang. Stillother essential oils known to those of skill in the art are alsocontemplated as being useful within the context of the present invention(e.g., International Cosmetic Ingredient Dictionary, 10^(th) and 12^(th)editions, 2004 and 2008, respectively, which are incorporated byreference).

In one embodiment, carvacrol and thymol-containing oils are purifiedfrom the species Origanum vulgare of a hirtum variety. Ideally this is ahybrid strain that produces high quality oils, but is not limited tothis genus, species or strain. The oil extract may also be obtained froma plant of the genus Nepeta including, but not limited to species Nepetaracemosa (catmint), Nepeta citriodora, Nepeta elliptica, Nepetahindostoma, Nepeta lanceolate, Nepeta leucophylla, Nepetalongiobracteata, Nepeta mussinii, Nepeta nepetella, Nepeta sibthorpii,Nepeta subsessilis, Nepeta tuberosa, Thymus glandulosus, Thymushyemalis, Thymus vulgaris and Thymus zygis.

As indicated above, isolates and/or derivatives of essential oils mayalso be employed in the present invention. For example, monoterpenephenols are particularly suitable for use in the present invention,which may be isolated and purified from plant oil extracts, or madesynthetically by known methods. Suitable monoterpene phenols mayinclude, for instance, thymol, carvacrol, eucalyptol, etc.

Thymol (isopropyl-cresol) is one particularly suitable monoterpenephenol, which is a crystalline substance that has a boiling point ofabout 238° C. at atmospheric pressure. Carvacrol (isopropyl-o-cresol),an isomer of thymol, is another suitable compound. Carvacrol is a liquidwith a boiling point of about 233° C. at atmospheric pressure. Thymoland carvacrol, as well as isomers thereof, may be derived from plant oilextracts or synthesized. For example, carvacrol may be synthesized bythe reaction of nitrous acid with 1-methyl-2-amino-4-propyl benzene. Inaddition to being employed in an isolated or pre-synthesized form,essential oils containing the monoterpene phenols as major constituentsmay be employed, with the final concentrations of the monoterpenephenols being within the ranges provided herein. The term “majorconstituent” generally refers to those essential oils having monoterpenephenols in an amount of more than 50 wt. %. It is well-known in the artthat such essential oils may also contain lesser amounts of otherconstituents, such as non-aromatic terpene compounds. Essential oilswith organic phenolic compounds as the major constituent include, forexample, anise oil, bay oil terpineless, clove bud, clove leaf, cloveoil, clove stem, origanum oil, Peru balsam, pimento oil, eucalyptus oil,and thyme oil.

Due to the stability achieved by the antimicrobial composition of thepresent invention, a relatively small amount of botanical oils may beemployed and still achieve the desired antimicrobial efficacy. Moreparticularly, the composition may employ botanical oils in an amount offrom about 0.1 wt. % to about 30 wt. %, in some embodiments from about0.5 wt. % to about 20 wt. %, and in some embodiments, from about 1 wt. %to about 10 wt. %.

B. Protein

The antimicrobial composition of the present invention also contains aprotein. Because the botanical oil tends to leach out during storage andbefore it is used in the desired application, the protein helps enhancethe long term stability of the oil and, in turn, antimicrobial efficacy.Without intending to be limited by theory, it is believed that thephysical structure of the protein can effectively encapsulate thebotanical oil and inhibit its premature release. Nevertheless, when itis desired to release the botanical oil prior to and/or during use, theprotein can disperse (e.g., disintegrate, dissolve, change physicalform, etc.) when placed in an aqueous environment. The amount of timeneeded for dispersal of the protein so that it releases the desiredantimicrobial active will depend at least in part upon the particularend-use design criteria. In most embodiments, the protein will begin todisperse and release the antimicrobial active within about 5 minutes,suitably within about 1 minute, more suitably within about 30 seconds,and most suitably within about 10 seconds.

Examples of suitable proteins include vegetable proteins, dairyproteins, animal proteins, as well as concentrates or isolates thereof.The protein source may be, for instance, milk (e.g., casein orcaeseinates), whey, corn (e.g., zein), wheat (e.g., wheat gluten), soy,or other vegetable or animal sources. Plant proteins are particularlysuitable for use in the present invention, such as zein, corn gluten,wheat gluten, whey protein, soy protein, etc. Any form of protein may beused, such as isolates, concentrates and flour. For example, soyproteins may be in the form of an isolate containing from about 75 wt %to about 98 wt. % protein, a concentrate containing from about 50 wt. %to about 75 wt. % protein, or flour containing from about 30 wt. % toabout 50 wt. % protein. In certain embodiments, it is desirable to use aprotein that is relatively pure, such as those having a protein contentof about 75 wt. % or more, and in some cases, about 85 wt. % or more.Gluten proteins, for instance, may be purified by washing away anyassociated starch to leave a composite of gliadin and glutenin proteins.In one particular embodiment, a vital wheat gluten is employed. Suchvital wheat gluten is commercially available as a creamy-tan powderproduced from wheat flour by drying freshly washed gluten. For instance,vital wheat gluten can be obtained from Archer Daniels Midland (“ADM”)of Decatur, Ill. under the designations WhetPro® 75 or 80. Similarly,purified soy protein isolates may be prepared by alkaline extraction ofa defatted meal and acid precipitation, a technique well-known and usedroutinely in the art. Such purified soy proteins are commerciallyavailable from ADM under the designation PRO-FAM®, which typically havea protein content of 90 wt. % or more. Other purified soy proteinproducts are also available from DuPont of Louisville, Ky. under thedesignation PRO-COTE® and from Central Soya under the designation PromieR.

If desired, the protein may also be modified using techniques known inthe art to improve its ability to disperse in an aqueous solution, whichmay be applied to the composition to release the botanical oil duringand/or just prior to use as described in more detail below. Suitablemodification techniques may include pH modification, denaturation,hydrolysis, acylation, reduction, oxidation, etc. Just as an example,gluten may sometimes absorb water until it begins to repel excess water.This results in gluten molecules that are associated closely togethersuch that they resist dispersion in aqueous solutions. To counteractthis tendency, the protein may be treated with a pH modifier to increaseits solubility in aqueous environments. Typically, the pH modifier is abasic reagent that can raise the pH of the protein, thereby causing itto become more soluble in aqueous solutions. Monovalentcation-containing basic reagents (hereafter “monovalent basic reagents”)are particularly suitable for use in the present invention. Examples ofsuch monovalent basic reagents include, for instance, alkali metalhydroxides (e.g., sodium hydroxide, ammonium hydroxide, etc.), ammonia,etc. Of course, multivalent reagents, such as alkaline metal hydroxides(e.g., calcium hydroxide) and alkaline metal oxides (e.g., calciumoxide), may also be employed if desired. When employed, the pH modifiermay be present in an amount such that the pH of the protein is fromabout 7 to about 14, and in some embodiments, from about 8 to about 12.

Hydrolysis of the protein material may also improve water solubility,and can be effected by treating the protein with a hydrolytic enzyme.Many enzymes are known in the art which hydrolyze protein materials,including, but not limited to, proteases, pectinases, lactases, andchymotrypsin. Enzyme hydrolysis is effected by adding a sufficientamount of enzyme to an aqueous dispersion of protein material, typicallyfrom about 0.1% to about 10% enzyme by weight of the protein material,and treating the enzyme and protein dispersion. After sufficienthydrolysis has occurred the enzyme may be deactivated by heating, andthe protein material may be precipitated from the solution by adjustingthe pH of the solution to about the isoelectric point of the proteinmaterial.

The antimicrobial composition of the present invention typically employsproteins in an amount of from about 30 wt. % to about 95 wt. %, in someembodiments from about 40 wt. % to about 90 wt. %, and in someembodiments, from about 50 wt. % to about 80 wt. %.

C. Carrier Fluid

A carrier fluid may also be employed in the antimicrobial composition tohelp render the protein more flowable under melt processing conditionsand able to receive the botanical oil within its internal structure.Suitable carrier fluids may include, for instance, polyhydric alcohols,such as sugars (e.g., glucose, sucrose, fructose, raffinose,maltodextrose, galactose, xylose, maltose, lactose, mannose, anderythrose), sugar alcohols (e.g., erythritol, xylitol, malitol,mannitol, and sorbitol), polyols (e.g., ethylene glycol, glycerol,propylene glycol, dipropylene glycol, butylene glycol, and hexanetrial), etc. Also suitable are hydrogen bond forming organic compoundswhich do not have hydroxyl group, including urea and urea derivatives;anhydrides of sugar alcohols such as sorbitan; animal proteins such asgelatin; vegetable proteins such as sunflower protein, soybean proteins,cotton seed proteins; and mixtures thereof. Other suitable carrierfluids may include phthalate esters, dimethyl and diethylsuccinate andrelated esters, glycerol triacetate, glycerol mono and diacetates,glycerol mono, di, and tripropionates, butanoates, stearates, lacticacid esters, citric acid esters, adipic acid esters, stearic acidesters, oleic acid esters, and other acid esters. Aliphatic carboxylicacids may also be used, such as lactic acid, maleic acid, acrylic acid,copolymers of ethylene and acrylic acid, polyethylene grafted withmaleic acid, polybutadiene-co-acrylic acid, polybutadiene-co-maleicacid, polypropylene-co-acrylic acid, polypropylene-co-maleic acid, andother hydrocarbon based acids. A low molecular weight carrier fluid ispreferred, such as less than about 20,000 g/mol, preferably less thanabout 5,000 g/mol and more preferably less than about 1,000 g/mol.

If desired, the carrier fluid may be selected to have a certain pH(refers to the pH prior to incorporation into the antimicrobialcomposition). For example, carrier fluids having a relatively low pH canreduce the tendency of gluten proteins to aggregate during meltprocessing. Thus, when gluten proteins are employed, a carrier fluid maybe selected that has a pH of about 6 or less, in some embodiments fromabout 1 to about 5, and in some embodiments, from about 2 to about 4.Examples of such carrier fluids may include aliphatic carboxylic acids,such as lactic acid, maleic acid, acrylic acid, etc. In otherembodiments, it may be desirable to use carrier fluids having a higherpH, such as when the plant protein is not generally sensitive to pH. Forexample, soy proteins generally lack the cysteine residues that lead toaggregation in gluten proteins. Thus, when employed, the soy protein maybe used with carrier fluids having a relatively wide range of pH levels.One example of such a carrier fluid is glycerol, which has a pH of about6.

The amount of the carrier fluids employed depends in part on the natureof the selected botanical oil and protein, but is typically from about 1wt. % to about 50 wt. %, in some embodiments from about 5 wt. % to about30 wt. %, and in some embodiments, from about 10 wt. % to about 20 wt.%.

D. Other Components

In addition to those noted above, still other additives may also beincorporated into the composition. For example, starch polymers, whichare often found in commercially available protein compositions, may alsobe employed in the present invention. When employed, such starchpolymers typically constitute from about 1 wt. % to about 50 wt. % ofthe composition, in some embodiments from about 10 wt. % to about 45 wt.%, and in some embodiments, from about 20 wt. % to about 40 wt. % of thecomposition.

Although starch polymers are produced in many plants, typical sourcesincludes seeds of cereal grains, such as corn, waxy corn, wheat,sorghum, rice, and waxy rice; tubers, such as potatoes; roots, such astapioca (i.e., cassava and manioc), sweet potato, and arrowroot; and thepith of the sago palm. Chemically modified starches may also be employedas they typically possess a higher degree of water sensitivity, andtherefore can help facilitate water sensitivity during use. Suchchemically modified starches may be obtained through typical processesknown in the art (e.g., esterification, etherification, oxidation, acidhydrolysis, enzymatic hydrolysis, etc.). Starch ethers and/or esters maybe particularly desirable, such as hydroxyalkyl starches, carboxymethylstarches, etc. The hydroxyalkyl group of hydroxylalkyl starches maycontain, for instance, 2 to 10 carbon atoms, in some embodiments from 2to 6 carbon atoms, and in some embodiments, from 2 to 4 carbon atoms.Representative hydroxyalkyl starches such as hydroxyethyl starch,hydroxypropyl starch, hydroxybutyl starch, and derivatives thereof.Starch esters, for instance, may be prepared using a wide variety ofanhydrides (e.g., acetic, propionic, butyric, and so forth), organicacids, acid chlorides, or other esterification reagents. The degree ofesterification may vary as desired, such as from 1 to 3 ester groups perglucosidic unit of the starch. The starch polymer may contain differentweight percentages of amylose and amylopectin, different polymermolecular weights, etc. High amylose starches contain greater than about50% by weight amylase and low amylose starches contain less than about50% by weight amylose. Although not required, low amylose starcheshaving an amylose content of from about 10% to about 40% by weight, andin some embodiments, from about 15% to about 35% by weight, areparticularly suitable for use in the present invention. Examples of suchlow amylose starches include corn starch and potato starch, both ofwhich have an amylose content of approximately 20% by weight.

Dispersion aids may also be employed to help create a uniform dispersionof the oil/protein/carrier fluid and retard or prevent separation of theantimicrobial composition into constituent phases. When employed, thedispersion aid(s) typically constitute from about 0.01 wt. % to about 10wt. %, in some embodiments from about 0.1 wt. % to about 5 wt. %, and insome embodiments, from about 0.5 wt. % to about 4 wt. % of theantimicrobial composition. Although any dispersion aid may generally beemployed in the present invention, surfactants having a certainhydrophilic/lipophilic balance may improve the long-term stability ofthe composition. As is known in the art, the relative hydrophilicity orlipophilicity of an emulsifier can be characterized by thehydrophilic/lipophilic balance (“HLB”) scale, which measures the balancebetween the hydrophilic and lipophilic solution tendencies of acompound. The HLB scale ranges from 0.5 to approximately 20, with thelower numbers representing highly lipophilic tendencies and the highernumbers representing highly hydrophilic tendencies. In some embodimentsof the present invention, the HLB value of the surfactants is from about1 to about 15, in some embodiments from about 1 to about 12 and in someembodiments, from about 2 to about 10. If desired, two or moresurfactants may be employed that have HLB values either below or abovethe desired value, but together have an average HLB value within thedesired range.

One particularly suitable class of surfactants for use in the presentinvention are nonionic surfactants, which typically have a hydrophobicbase (e.g., long chain alkyl group or an alkylated aryl group) and ahydrophilic chain (e.g., chain containing ethoxy and/or propoxymoieties). For instance, some suitable nonionic surfactants that may beused include, but are not limited to, ethoxylated alkylphenols,ethoxylated and propoxylated fatty alcohols, polyethylene glycol ethersof methyl glucose, polyethylene glycol ethers of sorbitol, ethyleneoxide-propylene oxide block copolymers, ethoxylated esters of fatty(C₈-C₁₈) acids, condensation products of ethylene oxide with long chainamines or amides, condensation products of ethylene oxide with alcohols,fatty acid esters, monoglyceride or diglycerides of long chain alcohols,and mixtures thereof. In one particular embodiment, the nonionicsurfactant may be a fatty acid ester, such as a sucrose fatty acidester, glycerol fatty acid ester, propylene glycol fatty acid ester,sorbitan fatty acid ester, pentaerythritol fatty acid ester, sorbitolfatty acid ester, and so forth. The fatty acid used to form such estersmay be saturated or unsaturated, substituted or unsubstituted, and maycontain from 6 to 22 carbon atoms, in some embodiments from 8 to 18carbon atoms, and in some embodiments, from 12 to 14 carbon atoms. Inone particular embodiment, mono- and di-glycerides of fatty acids may beemployed in the present invention.

The composition may also contain a preservative or preservative systemto inhibit the growth of microorganisms over an extended period of time.Suitable preservatives may include, for instance, alkanols, disodiumEDTA (ethylenediamine tetraacetate), EDTA salts, EDTA fatty acidconjugates, isothiazolinone, benzoic esters (parabens) (e.g.,methylparaben, propylparaben, butylparaben, ethylparaben,isopropylparaben, isobutylparaben, benzylparaben, sodium methylparaben,and sodium propylparaben), benzoic acid, propylene glycols, sorbates,urea derivatives (e.g., diazolindinyl urea), and so forth. Othersuitable preservatives include those sold by Sutton Labs, such as“Germall 115” (amidazolidinyl urea), “Germall II” (diazolidinyl urea),and “Germall Plus” (diazolidinyl urea and iodopropynyl butylcarbonate).Another suitable preservative is Kathon CG®, which is a mixture ofmethylchloroisothiazolinone and methylisothiazolinone available fromRohm & Haas; Mackstat H 66 (available from McIntyre Group, Chicago,Ill.). Still another suitable preservative system is a combination of56% propylene glycol, 30% diazolidinyl urea, 11% methylparaben, and 3%propylparaben available under the name GERMABEN® II from InternationalSpecialty Products of Wayne, N.J.

To better enhance the benefits to consumers, other optional ingredientsmay also be used. For instance, some classes of ingredients that may beused include, but are not limited to: antioxidants (product integrity);anti-reddening agents, such as aloe extract; astringents-cosmetic(induce a tightening or tingling sensation on skin); colorants (impartcolor to the product); deodorants (reduce or eliminate unpleasant odorand protect against the formation of malodor on body surfaces);fragrances (consumer appeal); opacifiers (reduce the clarity ortransparent appearance of the product); skin conditioning agents; skinexfoliating agents (ingredients that increase the rate of skin cellturnover such as alpha hydroxy acids and beta hydroxyacids); skinprotectants (a drug product which protects injured or exposed skin ormucous membrane surface from harmful or annoying stimuli); andthickeners (to increase viscosity).

While a wide variety of different components may be employed, it istypically desired that the antimicrobial composition is formed withoutthe use of solvents, particularly organic solvents, such as organicalcohols (e.g., ethanol). Not only does this enhance manufacturingefficiency, but it also limits the evaporation of the botanical oil thatmight otherwise be encountered during removal of the solvent. While thecomposition may be generally free of such solvents, it should of coursebe understood that a small amount may still be present in the resultingcomposition. Regardless, the composition typically contains solvents inan amount less than about 20 wt. %, in some embodiments less than about10 wt. %, and in some embodiments, from about 0.01 wt. % to about 5 wt.%.

II. Melt Processing Technique

As indicated above, the antimicrobial composition of the presentinvention is formed by processing the components together in a meltblending device (e.g., extruder). The mechanical shear and heat providedby the device allows the components to be blended together in a highlyefficient manner without the use of a solvent. Batch and/or continuousmelt blending techniques may be employed in the present invention. Forexample, a mixer/kneader, Banbury mixer, Farrel continuous mixer,single-screw extruder, twin-screw extruder, roll mill, etc., may beutilized. One particularly suitable melt-blending device is aco-rotating, twin-screw extruder (e.g., USALAB twin-screw extruderavailable from Thermo Electron Corporation of Stone, England or anextruder available from Werner-Pfleiderer from Coperion Ramsey, N.J.).The raw materials (e.g., botanical oil, protein, carrier fluid, etc.)may be supplied to the melt blending device separately and/or as ablend. For example, the protein and/or botanical oil may be initiallyfed to a feeding port of the twin-screw extruder. Thereafter, a carrierfluid may be injected into the extruder downstream from the botanicaloil and protein. Alternatively, the components may be simultaneously fedto the feed throat of the extruder or separately at a different pointalong its length.

Regardless, the materials are dispersively blended under lowshear/pressure and at a low temperature to minimize protein dissociationassociated with aggregation. Nevertheless, the temperature is stilltypically slightly at or above the softening point of the protein. Forexample, melt blending typically occurs at a temperature of from about20° C. to about 100° C., in some embodiments, from about 30° C. to about80° C., and in some embodiments, from about 40° C. to about 70° C.Likewise, the apparent shear rate during melt blending may range fromabout 100 seconds⁻¹ to about 5,000 seconds⁻¹, in some embodiments fromabout 200 seconds⁻¹ to about 2,000 seconds⁻¹, and in some embodiments,from about 400 seconds⁻¹ to about 1,200 seconds⁻¹. The apparent shearrate is equal to 4Q/πR³, where Q is the volumetric flow rate (“m³/s”) ofthe polymer melt and R is the radius (“m”) of the capillary (e.g.,extruder die) through which the melted polymer flows. The apparent meltviscosity of the resulting antimicrobial composition may be relativelylow, such as from about 1 to about 100 Pascal seconds (Pa·s), in someembodiments from about 5 to about 60 Pa·s, and in some embodiments, fromabout 20 to about 50 Pa·s, as determined at a temperature of 160° C. anda shear rate of 1000 sec⁻¹. The melt flow index (190° C., 2.16 kg) ofthe composition may also range from about 0.05 to about 50 grams per 10minutes, in some embodiments from about 0.1 to about 15 grams per 10minutes, and in some embodiments, from about 0.5 to about 5 grams per 10minutes.

Once formed, the antimicrobial composition of the present invention maybe used in a variety of forms, such as particles, lotion, cream, jelly,liniment, ointment, salve, oil, foam, gel, film, wash, coating, liquid,capsule, tablet, concentrate, etc. In one particular embodiment, forexample, the antimicrobial composition may be formed into a film, eitheralone or in conjunction with an additional film-forming material. Thefilm may be used in a wide variety of applications, such as in thepackaging of items (e.g., food products, medical products, garments,garbage, absorbent articles (e.g., diapers), etc. The film may have amono-layered or multi-layered structure. Multilayer films normallycontain at least one base layer and at least one skin layer, but maycontain any number of layers desired. The base layer and/or the skinlayer may contain the antimicrobial composition of the presentinvention. Any known technique may be used to form a film from thecompounded material, including blowing, casting, flat die extruding,etc. In one particular embodiment, the film may be formed by a blownprocess in which a gas (e.g., air) is used to expand a bubble of theextruded polymer blend through an annular die. The bubble is thencollapsed and collected in flat film form. Processes for producing blownfilms are described, for instance, in U.S. Pat. No. 3,354,506 to Raley;U.S. Pat. No. 3,650,649 to Schippers; and U.S. Pat. No. 3,801,429 toSchrenk et al., as well as U.S. Patent Application Publication Nos.2005/0245162 to McCormack, et al. and 2003/0068951 to Bows, et al., allof which are incorporated herein in their entirety by reference theretofor all purposes. In yet another embodiment, however, the film is formedusing a casting technique.

Besides being formed into a film, the antimicrobial composition of thepresent invention may also be formed into particles and applied to othertypes of articles. Powderization may be accomplished using any of avariety of known techniques. Suitable pulverizing techniques mayinclude, for instance, cryogenic disk mill or hammer mill, solid stateshear pulverization using cold extrusion technology, double stream mills(e.g., Type PSKM or PPSM mills available from Pallmann Industries), andother known powderization methods. Cryogenic downsizing techniques orcold extrusion pulverization techniques may be particularly suitable assuch techniques limit the degree to which the volatile botanical oil isheated and lost during powder formation. Examples of such techniques aredescribed in more detail, for instance, in U.S. Pat. No. 5,395,055 toShutov, et al., which is incorporated herein in its entirety byreference thereto for all purposes. The shape of the particles may varyas desired, such as spherical, nodular, flake, etc. The average size ofthe particles may also be selected to optimize the ability of thebotanical oil to be released during use. More particularly, the presentinventors have discovered that smaller particle sizes can generallyresult in a greater release rate of the oil when dispersed in an aqueoussolution due to their high surface area to volume ratio. However, at toosmall of a size, the botanical oil may become unstable during storageand actually begin to leach out of the particles prior to use. In thisregard, the present inventors have discovered that an average size offrom about 10 to about 3,000 micrometers, in some embodiments from about50 to about 800 micrometers, and in some embodiments, from about 100 toabout 600 micrometers, can help achieve a good balance between stabilityand releasibility.

Regardless of its particular form, the antimicrobial particles may beapplied to a wide variety of different articles for impartingantimicrobial efficacy. In one particular embodiment, the composition isapplied to a wipe. Such wipes may be used to reduce microbial or viralpopulations on a hard surface (e.g., sink, table, counter, sign, and soforth) or surface on a user/patient (e.g., skin, mucosal membrane, suchas in the mouth, nasal passage, stomach, vagina, etc., wound site,surgical site, and so forth). The wipe may provide an increased surfacearea to facilitate contact of the composition with microorganisms. Inaddition, the wipe may also serve other purposes, such as providingwater absorption, barrier properties, etc. The wipe may also eliminatemicroorganisms through frictional forces imparted to the surface.

The wipe may be formed from any of a variety of materials as is wellknown in the art. Typically, however, the wipe includes a fibrous webthat contains absorbent fibers. For example, the wipe may be a paperproduct containing one or more paper webs, such as facial tissue, bathtissue, paper towels, napkins, and so forth. The paper product may besingle-ply in which the web forming the product includes a single layeror is stratified (i.e., has multiple layers), or multi-ply, in which thewebs forming the product may themselves be either single ormulti-layered. Normally, the basis weight of such a paper product isless than about 120 grams per square meter (“gsm”), in some embodimentsless than about 80 gsm, in some embodiments less than about 60 grams persquare meter, and in some embodiments, from about 10 to about 60 gsm.Any of a variety of materials can also be used to form the paper web(s)of the product. For example, the material used to make the paper productmay include absorbent fibers formed by a variety of pulping processes,such as kraft pulp, sulfite pulp, thermomechanical pulp, etc. The pulpfibers may include softwood fibers having an average fiber length ofgreater than 1 mm and particularly from about 2 to 5 mm based on alength-weighted average. Such softwood fibers can include, but are notlimited to, northern softwood, southern softwood, redwood, red cedar,hemlock, pine (e.g., southern pines), spruce (e.g., black spruce),combinations thereof, and so forth. Exemplary commercially availablepulp fibers suitable for the present invention include those availablefrom Kimberly-Clark Corporation under the trade designations“Longlac-19”. Hardwood fibers, such as eucalyptus, maple, birch, aspen,and so forth, can also be used. In certain instances, eucalyptus fibersmay be particularly desired to increase the softness of the web.Eucalyptus fibers can also enhance the brightness, increase the opacity,and change the pore structure of the web to increase its wickingability. Moreover, if desired, secondary fibers obtained from recycledmaterials may be used, such as fiber pulp from sources such as, forexample, newsprint, reclaimed paperboard, and office waste. Further,other natural fibers can also be used in the present invention, such asabaca, sabai grass, milkweed floss, pineapple leaf, bamboo, algae, andso forth. In addition, in some instances, synthetic fibers can also beutilized.

If desired, the absorbent fibers (e.g., pulp fibers) may be integratedwith synthetic fibers to form a composite. Synthetic thermoplasticfibers may also be employed in the nonwoven web, such as those formedfrom polyolefins, e.g., polyethylene, polypropylene, polybutylene, etc.;polytetrafluoroethylene; polyesters, e.g., polyethylene terephthalateand so forth; polyvinyl acetate; polyvinyl chloride acetate; polyvinylbutyral; acrylic resins, e.g., polyacrylate, polymethylacrylate,polymethylmethacrylate, and so forth; polyamides, e.g., nylon; polyvinylchloride; polyvinylidene chloride; polystyrene; polyvinyl alcohol;polyurethanes; polylactic acid; polyhydroxyalkanoate; copolymersthereof; and so forth. Because many synthetic thermoplastic fibers areinherently hydrophobic (i.e., non-wettable), such fibers may optionallybe rendered more hydrophilic (i.e., wettable) by treatment with asurfactant solution before, during, and/or after web formation. Otherknown methods for increasing wettability may also be employed, such asdescribed in U.S. Pat. No. 5,057,361 to Sayovitz, et al., which isincorporated herein in its entirety by reference thereto for allpurposes. The relative percentages of such fibers may vary over a widerange depending on the desired characteristics of the composite. Forexample, the composite may contain from about 1 wt. % to about 60 wt. %,in some embodiments from 5 wt. % to about 50 wt. %, and in someembodiments, from about 10 wt. % to about 40 wt. % synthetic polymericfibers. The composite may likewise contain from about 40 wt. % to about99 wt. %, in some embodiments from 50 wt. % to about 95 wt. %, and insome embodiments, from about 60 wt. % to about 90 wt. % absorbentfibers.

Composites, such as described above, may be formed using a variety ofknown techniques. For example, a nonwoven composite may be formed thatis a “coform material” that contains a mixture or stabilized matrix ofthermoplastic fibers and a second non-thermoplastic material. As anexample, coform materials may be made by a process in which at least onemeltblown die head is arranged near a chute through which othermaterials are added to the web while it is forming. Such other materialsmay include, but are not limited to, fibrous organic materials such aswoody or non-woody pulp such as cotton, rayon, recycled paper, pulpfluff and also superabsorbent particles, inorganic and/or organicabsorbent materials, treated polymeric staple fibers and so forth. Someexamples of such coform materials are disclosed in U.S. Pat. Nos.4,100,324 to Anderson, et al.; 5,284,703 to Everhart, et al.; and5,350,624 to Georger, et al.; which are incorporated herein in theirentirety by reference thereto for all purposes. Alternatively, thenonwoven composite may be formed be formed by hydraulically entanglingstaple length fibers and/or filaments with high-pressure jet streams ofwater. Various techniques for hydraulically entangling fibers aregenerally are disclosed, for example, in U.S. Pat. Nos. 3,494,821 toEvans and 4,144,370 to Bouolton, which are incorporated herein in theirentirety by reference thereto for all purposes. Hydraulically entanglednonwoven composites of continuous filaments (e.g., spunbond web) andnatural fibers (e.g., pulp) are disclosed, for example, in U.S. Pat.Nos. 5,284,703 to Everhart, et al. and 6,315,864 to Anderson, et al.,which are incorporated herein in their entirety by reference thereto forall purposes. Hydraulically entangled nonwoven composite of staple fiberblends (e.g., polyester and rayon) and natural fibers (e.g., pulp), alsoknown as “spunlaced” fabrics, are described, for example, in U.S. Pat.No. 5,240,764 to Haid, et al., which is incorporated herein in itsentirety by reference thereto for all purposes.

Regardless of the materials or processes utilized to form the wipe, thebasis weight of the wipe is typically from about 20 to about 200 gramsper square meter (“gsm”), and in some embodiments, between about 35 toabout 100 gsm. Lower basis weight products may be particularly wellsuited for use as light duty wipes, while higher basis weight productsmay be better adapted for use as industrial wipes.

The wipe may assume a variety of shapes, including but not limited to,generally circular, oval, square, rectangular, or irregularly shaped.Each individual wipe may be arranged in a folded configuration andstacked one on top of the other to provide a stack of wet wipes. Suchfolded configurations are well known to those skilled in the art andinclude c-folded, z-folded, quarter-folded configurations and so forth.For example, the wipe may have an unfolded length of from about 2.0 toabout 80.0 centimeters, and in some embodiments, from about 10.0 toabout 25.0 centimeters. The wipes may likewise have an unfolded width offrom about 2.0 to about 80.0 centimeters, and in some embodiments, fromabout 10.0 to about 25.0 centimeters. The stack of folded wipes may beplaced in the interior of a container, such as a plastic tub, to providea package of wipes for eventual sale to the consumer. Alternatively, thewipes may include a continuous strip of material which has perforationsbetween each wipe and which may be arranged in a stack or wound into aroll for dispensing. Various suitable dispensers, containers, andsystems for delivering wipes are described in U.S. Pat. Nos. 5,785,179to Buczwinski, et al.; 5,964,351 to Zander; 6,030,331 to Zander;6,158,614 to Haynes, et al.; 6,269,969 to Huang, et al.; 6,269,970 toHuang, et al.; and 6,273,359 to Newman, et al., which are incorporatedherein in their entirety by reference thereto for all purposes.

The composition may be incorporated into the wipe in a variety ofdifferent ways. For example, the composition may be applied to a surfaceof the wipe using known techniques, such as printing, dipping, spraying,melt extruding, coating (e.g., solvent coating, powder coating, brushcoating, etc.), foaming, and so forth. If desired, the composition maybe applied in a pattern that covers from about 5% to about 95%, in someembodiments from about 10% to about 90%, and in some embodiments, fromabout 20% to about 75% of a surface of the wipe. Such patternedapplication may have various benefits, including enhanced aestheticappeal, improved absorbency, etc. The particular type or style of thepattern is not a limiting factor of the invention, and may include, forexample, any arrangement of stripes, bands, dots, or other geometricshape. The pattern may include indicia (e.g., trademarks, text, andlogos), floral designs, abstract designs, any configuration of artwork,etc. It should be appreciated that the “pattern” may take on virtuallyany desired appearance. The composition may also be blended with thefibers used to form the wipe. This may be particularly useful when thecomposition is in the form of particles. For example, such particles maybe blended with the absorbent fibers (e.g., pulp fibers, staple fibers,etc.) during hydraulic entanglement, coforming, etc. The particles mayalso be incorporated into the thermoplastic material of the wipe (e.g.,meltblown web) using known techniques.

The amount of the antimicrobial composition on the wipe may varydepending on the nature of the substrate and its intended application.For example, the add-on level of the composition may be from about 5% toabout 100%, in some embodiments from about 10% to about 80%, and in someembodiments, from about 20% to about 70%. The “add-on level” isdetermined by subtracting the weight of the untreated substrate from theweight of the treated substrate, dividing this calculated weight by theweight of the untreated substrate, and then multiplying by 100%. Loweradd-on levels may provide optimum functionality of the substrate, whilehigher add-on levels may provide optimum antimicrobial efficacy.

To use the composition, an aqueous solution may simply be added, therebydispersing the protein and releasing the botanical oil. The aqueoussolution may contain only water, or it may contain water in combinationwith other components. For example, a weak acid may be employed to helpdisperse the protein and facilitate the release of the oil upon contactwith the aqueous solution. Suitable acids for this purpose may include,for instance, organic carboxylic acids, such as citric acid, oxalicacid, lactic acid, acetic acid, etc. Regardless, the present inventorshave surprisingly discovered that the amount of the botanical oilreleased into the aqueous solution can be even greater than the normalsolubility limit of the oil in water. Without intending to be limited bytheory, it is believed that this can be achieved because the physicalstructure of the protein is able to effectively “carry” the volatileinto the released solution. For example, the solubility limit of thymolin water (at 25° C.) is typically about 0.1 wt. %. When released fromthe composition of the present invention, however, the concentration ofthymol in the released solution can be greater than 0.1 wt. %, in someembodiments greater than about 0.5 wt. %, in some embodiments from about1 wt. % to about 10 wt. %, and in some embodiments, from about 2 wt. %to about 8 wt. %.

The present inventors have discovered that the composition of thepresent invention may inhibit (e.g., reduce by a measurable amount or toprevent entirely) the growth of one or more microorganisms when exposedthereof. Examples of microorganisms that may be inhibited includebacteria, protozoa, algae, and fungi (e.g., molds and yeast).Furthermore is possible to use this invention to inactivate viruses,prions and other infectious particles. For example, the composition mayinhibit the growth of several medically significant bacteria groups,such as Gram negative rods (e.g., Entereobacteria); Gram negative curvedrods (e.g., Heliobacter, Campylobacter, etc.); Gram negative cocci(e.g., Neisseria); Gram positive rods (e.g., Bacillus, Clostridium,etc.); Gram positive cocci (e.g., Staphylococcus, Streptococcus, etc.);obligate intracellular parasites (e.g., Ricckettsia and Chlamydia); acidfast rods (e.g., Myobacterium, Nocardia, etc.); spirochetes (e.g.,Treponema, Borellia, etc.); and mycoplasmas (i.e., tiny bacteria thatlack a cell wall). Particularly species of bacteria that may beinhibited with the composition of the present invention includeEscherichia coli (Gram negative rod), Klebsiella pneumonia (Gramnegative rod), Streptococcus (Gram positive cocci), Salmonellacholeraesuis (Gram negative rod), Staphyloccus aureus (Gram positivecocci), and P. aeruginosa (Gram negative rod). In addition to bacteria,other microorganisms of interest include fungi (e.g., Aspergillus niger)and yeasts (e.g., Candida albicans).

Upon exposure for a certain period of time, the composition may providea log reduction of at least about 2, in some embodiments at least about3, in some embodiments at least about 4, and in some embodiments, atleast about 5 (e.g., about 6). Log reduction, for example, may bedetermined from the % population killed by the composition according tothe following correlations:

% Reduction Log Reduction 90 1 99 2 99.9 3 99.99 4 99.999 5 99.9999 6

Such a log reduction may be achieved in accordance with the presentinvention after only a relatively short exposure time. For example, thedesired log reduction may be achieved after exposure for only 30minutes, in some embodiments 15 minutes, in some embodiments 10 minutes,in some embodiments 5 minutes, in some embodiments 1 minute, and in someembodiments, 30 seconds.

The present invention may be better understood with reference to thefollowing examples.

Materials Employed

-   -   Thymol (99.5% purity) was obtained from Sigma-Aldrich.    -   Carvacrol (98% purity) was obtained from Sigma-Aldrich.    -   Eucalyptol (C80601) was obtained from Sigma-Aldrich.    -   Soy Protein Flour (50% protein, 50% starch) was obtained from        ADM.    -   WhetPro® 75 vital wheat gluten (75% protein, 25% starch) was        obtained from ADM.    -   Emery 917 Glycerine (or Glycerol) was obtained from Cognis        Oleochemicals.    -   L-Lactic Acid (Purac)

Test Methods Thymol Stability

Samples were placed in an oven at 40° C., 50° C., or 55° C. for acertain number of days. The residual thymol level was determined through“High Performance Liquid Chromatography (HPLC) analysis.” Moreparticularly, the thymol level in each sample was determined bygenerating a thymol calibration curve by the following method.Approximately 70 mg thymol was weighed into a 100-mL volumetric flask.Approximately 50-mL of a 0.1% acetic acid:IPA mixture (50:50) was addedto the flask and the contents swirled to promote dissolution. The volumewas diluted with a 0.1% acetic acid:IPA mixture (50:50) and subsequentdilutions were performed to generate a calibration curve with aconcentration range of approximately 700 μg/mL to 70 μg/mL. Samples wereprepared as follows. Approximately 100 mg of sample was used for eachcode, where each code was analyzed in duplicate at every pull point. Themeasured material was cut up into small pieces and placed into a 40-mLvial. To each vial, 10.0 mL 0.1% acetic acid was added and the contentswere shaken and sonicated for 30 minutes periods until the sample isdispersed. To each vial, 10.0 mL IPA was added and the contents weresonicated for 10 minutes to promote mixing and extraction of thymol. Theresulting solutions were filtered through nylon filters prior toinjection. Thymol levels were calculated using the thymol calibrationcurve described above.

HPLC Equipment & Conditions Column: Phenomenex NH₂ Column TemperatureAmbient

Mobile Phase: 50:50 (IPA:0.1% acetic acid)Flow rate: 0.6 mL/min.Injection volume: 15 microlitersELS detection: 280 nm

Thymol Concentrations in Extractions

An aliquot of a sample was centrifuged at approximately 5000 rpm untilvisible settling occurred (approximately 30 minutes). The solution wasfiltered using two (2) different types of syringe filters: (1) Pall LifeAcrodisc 13 mm 0.2 micron nylon membrane and (2) Whatman Puradisc-0.2micron polyethersulfone membrane with polypropylene housing. 1.0 mL ofthe centrifuged solution was pipetted into a 10-mL flask. The contentswere dissolved and diluted with a 0.1% AA:IPA (50:50) solution tovolume. The solution was then filtered with Pall Acrodisc 0.45 micronnylon membrane. The thymol concentration was determined through “HighPerformance Liquid Chromatography (HPLC) analysis” according to thefollowing conditions:

HPLC Equipment & Conditions

HPLC: Agilent 1100 HPLC system.Column: Phenomenex Luna NH₂ (5 μm, 250 mm×4.6 mm)-ambient

Detector: UV/Vis at 280 nm

Mobile Phases: (75:25) (IPA:0.1% acetic acid)Flow Rate: approximately 0.6 mL/min.Injection Volume: approximately 15 μLRun Time: 6 minutes

Zone of Inhibition

To determine antimicrobial efficacy, a zone of inhibition test wasperformed. More specifically, a 0.05 g sample was placed on a freshlyspreading lawn of test microorganism on TSA (Trypticase Soy Agar). Twomicroorganisms were used, Staphylococcus aureus (ATCC #27660) as a Grampositive bacteria and

Escherichia coli as a Gram negative bacteria (ATCC #25922). After 24hours incubation at 37° C., plates were measured for clear zones ofinhibition surrounding each sample (Clear zone (mm)=diameter of clearzone−sample (wipe) diameter).

Microplate Assay

To determine the germicidal efficacy of an extracted thymol solution, amicroplate germicidal assay was performed. In this method, the testsolution was brought into contact with 60 wells of test microorganisms(4*106 CFU (colony forming unit)/well) coated on the bottom of 96 wellflat plates for 4½ minutes. At the end of the contact time, 200 μL of a“Letheen” neutralizing broth (included 0.5% Tween 80) was added to eachwell to deactivate the active ingredients. After addition of theneutralizer, 50 μL of TSB (Tryptic Soy Broth) was added and then themicroplate was incubated to allow for out-growth of survivors. Afterincubation, the number of wells showing growth of the targetmicroorganism was recorded. If the media in the well was turbid, thenthe well was counted as a failure to disinfect. If the well was notturbid after incubation, then the well was recorded as achievingdisinfection. All tests were performed against two differentmicroorganisms, Staphylococcus aureus (ATCC #6538) as Gram positivebacteria and Pseudomonas aeruginosa (ATCC #15442) as Gram negativebacteria.

Example 1

A “PRISM USALAB 16” lab scale twin screw extruder was employed to meltprocess five (5) different samples of protein (WhetPro® 75 or soyflour), glycerol, and thymol. The extruder contained eleven (11)different zones, although zones 1 through 5 were not utilized in thisExample. Temperature zone 11 was a strand die. The protein and thymolwere pre-blended (6.6 wt % thymol and 93.4%) and subsequently added tothe extruder at zone 6 at a feed rate of 0.5 lbs/hr. Glycerol was thenadded at zone 7 at a feed rate of 0.21 lb/hr. The screw configurationwas composed of conveying elements at zones 6 and 7, kneading blocks atzones 8 and 9, and conveying elements at zone 10. The screw speed was 50rpm. The resulting strand was pelletized to form a pellet with a size ofabout 3 mm in diameter. The temperature profile for each of the five (5)blend samples is set forth below.

Target Temperature Profile (° C.) Protein Temp. Zone Zone Zone Zone ZoneZone Sample Type (° C.) 6 7 8 9 10 11 1 Gluten Ambient 28 31 32 34 32 332 Gluten 50° C. 27 31 34 40 50 41 3 Gluten 90° C. 29 33 37 49 90 48 4Gluten 120° C.  35 42 51 68 120 67 5 Soy 90° C. 25 28 31 42 90 61 flour

After processing, it was determined that Samples 1-5 retained 90.8 wt.%, 88.2 wt. %, 88.4 wt. %, 85.4 wt. %, and 99.4 wt. %, respectively, oftheir initial thymol levels. Once formed, the samples were put into anair tight bag and placed in −10° C. freezer. The resulting samples weretested for thymol stability and zone of inhibition using the testmethods described above. The results are set forth below in Tables 1 and2.

TABLE 1 Thymol Level After Aging at 40° C. Thymol level (wt. %) Sample 1day 4 days 10 days 21 days 39 days 60 days 1 4.07 3.75 4.09 3.83 3.693.44 2 4.04 3.95 3.78 3.91 4.37 3.97 3 4.42 4.4 4.14 4.37 3.99 3.88 44.16 4.49 4.57 4.72 4.52 3.99 5 4.21 4.51 4.42 4.2 4.27 4.58

TABLE 2 Zone of Inhibition Area After Aging at 40° C. Area of ZOI (mm) 0days 1 day 4 days 10 days 20 days 40 days 60 days E. S. E. S. E. S. E.S. E. S. E. S. E. S. Sample coli aureus coli aureus coli aureus coliaureus coli aureus coli aureus coli aureus 1 5 6 6 6 7 6 6 5 7 4 6 4 6 42 5 6 6 6 6 6 6 5 7 4 7 4 6 4 3 6 6 6 6 6 6 5 5 7 4 6 3 5 4 4 5 6 6 6 65 5 4 6 5 5 3 5 4 5 10 10 10 10 10 11 5 3 2 2 1.5 2.5 0 0

As indicated in Table 1, the thymol levels remained high even afterapproximately 60 days of aging at 40° C. Table 2 also reveals that afterextrusion, thymol in Samples 1-4 still maintained antimicrobialactivity, even after 60 days of aging at 40° C. Example 5 (soy flour at120° C.) did not exhibit activity after 60 days, but was still effectiveafter 40 days. Without intending to be limited by theory, it is believedthat this difference may stem from the difference in proteinconcentrations between the WhetPro® 75 (75% protein) and the soy flour(50% protein).

SEM microphotographs were taken of Sample 3 and a control samplecontaining no thymol (70 wt. % gluten and 30 wt. % glycerol). Theresults are shown in FIGS. 1-4. As shown, there is no structural ormorphological difference between the sample containing thymol (Sample 3,FIGS. 3-4) and the control sample (FIGS. 1-2). This indicates thatthymol is homogenously compatible and retained within the proteinstructure. Also, there is no phase separation between the thymol andprotein/glycerol matrix, which helps to providing for the prolongedrelease of the thymol.

Example 2

WhetPro® 75 was placed in desiccant glass jar containing ammoniumhydroxide solution (20-35% ammonia, 65-80% water) for 10 days. Themixture was then extruded with thymol at a concentration of 85 wt. %treated gluten and 15 wt. % thymol. Specifically, a PRISM USALAB 16” labscale twin screw extruder was employed to melt process the ammoniumhydroxide treated WhetPro® 75 and thymol. The extruder contained eleven(11) different zones, although zones 1 through 5, and 11 were notutilized in this Example. The extruder was used without the die system(zone 11) to allow for ease of material to exit extruder. The treatedprotein and thymol were pre-blended (17 wt % thymol and 83 wt. %protein) and subsequently added to the extruder at zone 6 at a feed rateof 0.4 lbs/hr. The screw configuration was composed of conveyingelements at zones 6 and 7, kneading blocks at zones 8 and 9, andconveying elements at zone 10. The screw speed was 100 rpm. Thetemperature profile for zones 6-10 was 35° C., 44° C., 56° C., 70° C.,70° C. respectively.

The material was pelletized and cooled by placing in −32° C. for minimumof 24 hrs, resulting cooled material was powderized via Brickmann/Retschlab scale grinding mill (set speed=1) and collected at a size of 250 to425 μm by sieving. Sample was tested for thymol stability after aging at50° C. for 1, 4, 15, and 18 days using the test methods described above.The results indicated that when gluten was treated with NH₄OH, the rateloss of thymol decreased by approximately one half (0.01% per hour) ascompared to a control of untreated gluten (0.02% per hour).

Example 3

A “PRISM USALAB 16” lab scale twin screw extruder was employed to meltprocess WhetPro® 75, lactic acid, and thymol. The extruder containedeleven (11) different zones, although zones 1 through 5, and 11 were notutilized in this Example. The extruder was used without the die system(zone 11) to allow for ease of material to exit extruder. The proteinand thymol were pre-blended (17.3 wt % thymol) and subsequently added tothe extruder at zone 6 at a feed rate of 0.5 lbs/hr. Lactic acid wasthen added at zone 7 at a feed rate of 0.087 lb/hr to give a compositionof 70.4% WhetPro®, 14.8% lactic acid, 14.7% thymol. The screwconfiguration was composed of conveying elements at zones 6 and 7,kneading blocks at zones 8 and 9, and conveying elements at zone 10. Thescrew speed was 50 rpm. The temperature profile for zones 6-10 was 24°C., 32° C., 42° C., 70° C., 70° C. respectively. The resulting materialwas contained in plastic bag and stored at −32° C. Cooled material wasdownsized via Brickmann/Retsch lab scale grinding mill (set speed=1) andcollected at a size of 250 to 425 μm by sieving. The samples were testedfor thymol stability after aging at 50° C. for 1, 4, 15, 18, 56, 104days using the test methods described above. The results are set forthin Table 3.

TABLE 3 Thymol Level After Aging at 50° C. Thymol Level (wt %) 0 1 4 1518 56 104 Example days days days days days days days 3 14.3 14 12.4 9.18.2 7.55 7.09

Example 4

A “PRISM USALAB 16” lab scale twin screw extruder was employed to meltprocess WhetPro® 75 Gluten and thymol. The extruder contained eleven(11) different zones, although zones 1 through 5, and 11 were notutilized in this Example. The extruder used a 0.75-inch die system (zone11) to allow for ease of material to exit extruder. The protein andthymol were pre-blended (17 wt % thymol and 83% protein) andsubsequently added to the extruder at zone 6 at a feed rate of 0.5lbs/hr to give a composition of 83% WhetPro® and 17% thymol. The screwconfiguration was composed of conveying elements at zones 6 and 7,kneading blocks at zones 8 and 9, and conveying elements at zone 10. Thescrew speed was 50 rpm. The temperature profile for zones 6-11 was 37°C., 47° C., 60° C., 70° C., 70° C., and 70° C., respectively. Theresulting material was contained in plastic bag and stored at −32° C.Cooled material was downsized via Brickmann/Retsch lab scale grindingmill (set speed=1) and collected at a size <250 μm. The samples weretested for thymol stability after aging at 55° C. for 0, 14, 19 days.The results are set forth in Table 4.

TABLE 4 Thymol Level After Aging at 55° C. Thymol Level (wt %) Example 0days 7 days 19 days 4 14.3 14 12.4

100 milliliters of deionized water was then added to 5.88 grams of thenon-aged particles, as well as to 1 gram of neat thymol. Theconcentration of thymol in water was determined for samples extractedfor 2, 10, and 60 minutes by the method entitled “Thymol Concentrationsin Extractions.” The results are shown in FIG. 5. As indicated, theamount of thymol released into water from the thymol/protein particlewas approximately four (4) times greater than a sample extracted fromneat thymol.

Example 5

A “PRISM USALAB 16” lab scale twin screw extruder was employed to meltprocess two (2) different samples of WhetPro® 75 (gluten), glycerol, andvolatile oil (carvacrol or eucalyptol). The extruder contained eleven(11) different zones, although zones 1 through 5 were not utilized inthis Example. Temperature zone 11 was a strand die. WhetPro® 75 wasadded to the extruder at zone 6 via a drop feeder at a feed rate of 0.5lbs/hr. Glycerol and the volatile oil were then added via a syringe pumpat zone 7 at a feed rate of 0.24 lb/hr. The formulation was set tocontain 81 wt. % WhetPro® 75, 11 wt. % glycerol, and 5 wt. % of volatileoil. The screw configuration was composed of conveying elements at zones6 and 7, kneading blocks at zones 8 and 9, and conveying elements atzone 10. The screw speed was 100 rpm. The temperature profile for thesamples are set forth below.

Pellet Target Temperature Profile (° C.) Volatile Size Temp. Zone ZoneZone Zone Zone Zone Sample Oil (mm) (° C.) 6 7 8 9 10 11 7 Carvacrol 370 ~35 ~40 ~55 70 70 70 8 Eucalyptol 3 70 ~35 ~40 ~55 70 70 70

Once formed, the samples were put into an air tight bag and placed in−10° C. freezer. The resulting samples were tested for volatile oilstability using the test method described above. The results are setforth below in Table 5.

TABLE 5 Volatile Oil Level After Aging at 50° C. Volatile Oil level (wt.%) Sample 0 days 5 days 10 days 20 days 40 days 7 3.70 3.80 3.00 3.102.95 8 1.66 1.54 1.53 0.43 0.59

Example 6

A “PRISM USALAB 16” lab scale twin screw extruder was employed to meltprocess WhetPro® 75, glycerol, and thymol. The extruder contained eleven(11) different zones, although zones 1 through 5, and 11 were notutilized in this Example. The extruder was used with a 0.75-inch diesystem (zone 11) to allow for ease of material to exit extruder. Theprotein and thymol were pre-blended (17.3 wt % thymol) and subsequentlyadded to the extruder at zone 6 at a feed rate of 0.5 lbs/hr. Glycerolwas then added at zone 7 at a feed rate of 0.087 lb/hr check processrounds to give a approximate composition of 71% WhetPro®, 14% glycerol,15% thymol. The screw configuration was composed of conveying elementsat zones 6 and 7, kneading blocks at zones 8 and 9, and conveyingelements at zone 10. The screw speed was 50 rpm. The temperature profilefor zones 9-11 was 70° C. The resulting material was contained inplastic bag and stored at −32° C. Cooled material was downsized viaBrickmann/Retsch lab scale grinding mill (set speed=1) and collected ata size of: <250, 250-425, 425-710, 710-1000 μm by sieving. The resultingsamples were tested for thymol stability at 55° C. using the test methoddescribed above. The results are set forth below in Table 6.

TABLE 6 Rate Loss of Thymol for Various Particle Size Ranges % Thymol(w/w) Particle Size (microns) after 7 day aging Rate loss (%/day) <250*2.3 12.1 250-425 5.7 8.9 425-710 6.7 7.9  710-1000 8.0 6.7 *An examplecalculation for the “rate loss” is as follows: [(15% initial thymol −2.3% thymol aged) ÷ 15% initial thymol] ÷ 7 days * 100 = 12.1%/day

As indicated, the amount of thymol lost from the thymol/protein particlewas dependent on particle size in that the loss was significantlyincreased for particles having a smaller particle size.

Example 7

A composition of 71% WhetPro®, 14% glycerol, 15% thymol was prepared anddownsized as described in Example 6. Particle size ranges of <250,250-425, and >425 microns were collected via seiving, Once formed, 100milliliters of deionized water was added to 6.67 grams of the non-agedparticles. The concentration of thymol in water was determined after 10minutes of extraction in water by the method described above. Theresults are shown in Table 7.

TABLE 7 Thymol Released for Various Particle Sizes Particle Size(microns) % Thymol in water (% wt) <250 0.062 250-425 0.0296 >425 0.017

As indicated, the amount of thymol released into water from thethymol/protein particle was dependent on particle size in that theconcentration of thymol was increase for reduced particle sizes.

Example 8

A composition of 71% WhetPro®, 14% glycerol, 15% thymol was prepared anddownsized as described in Example 6. The particle size range of <250 wascollected via seiving. Once formed, 50 milliliters of deionized waterwas added to 6.67 grams of the non-aged particles. The concentration ofthymol in water was determined at 2, 10, and 60 minutes by the methoddescribed above giving thymol concentrations by weight of 0.058%,0.057%, 0.072% respectively.

Example 9

A composition of 71% WhetPro®, 14% glycerol, 15% thymol was prepared anddownsized as described in Example 6. The particle size range of <250 wascollected via seiving. Once formed, 100 milliliters of ethanol was addedto 6.67 grams of the non-aged particles. The concentration of thymol inethanol was determined at 2, 10, and 60 minutes by the method describedabove giving thymol concentrations by weight of 0.255, 0.260%, 0.292%respectively.

Example 10

A composition of 71% WhetPro®, 14% glycerol, 15% thymol was prepared byextrusion and downsized as described in Example 6. The particle sizerange of 250-425 collected via seiving. Once formed, 100 milliliters of7.7×10⁻⁶ M citric acid in deionized water was added to 6.67 grams of thenon-aged particles. The concentration of thymol in solution wasdetermined after extraction for 10 minutes by the method described abovegiving a thymol concentration by weight of 0.032%.

Example 11

A composition of 71% WhetPro®, 14% glycerol, 15% thymol was prepared byextrusion and downsized as described in Example 6. The particle sizerange of 250-425 was collected via seiving, Once formed, 100 millilitersof 7.5% citric acid by weight in deionized water was added to 6.67 gramsof the non-aged particles. The concentration of thymol in solution wasdetermined after extraction for 10 minutes by the method described abovegiving thymol concentration by weight of 0.046%.

Example 12

A composition of 81% WhetPro®, 14% glycerol, 5% thymol was prepared byextrusion and downsized as described in Example 6. The particle sizerange of <250 micron was collected via seiving. Once formed, 100milliliters of 0.1% aqueous acetic acid was added to 20 grams of thenon-aged particles. The concentration of thymol in acetic acid solutionwas determined after extraction for 2, 10, and 60 minutes by the methoddescribed above giving thymol concentrations by weight of 0.069%,0.080%, 0.083% respectively.

Example 13

Various compositions containing different thymol concentrations wereextruded and downsized to particle size of <250 microns. The resultingsamples were tested for thymol stability at 55° C. using the test methoddescribed above. The results are set forth in Table 8.

TABLE 8 Thymol Level After Aging at 55° C. Composition % Thymol ThymolLevel (wt %) Protein Carrier Fluid Add-On 0 days 7 days 19 days 71%Gluten 14% glycerol 15 14.3 2.3 1.3 81% Gluten 14% glycerol 5 4.6 3 2.685% Gluten 14% glycerol 1 1 0.7 0.7

Table 8 illustrates that thymol loss was dependent on thymolconcentration and time. For example, higher concentrations resulted in ahigher thymol loss. Likewise, a longer aging time resulted in a lowerrate loss of thymol. Table 9 summarizes the thymol rate loss for eachcomposition at each aging time interval of 0 days to 7 and 7 days to 19days.

TABLE 9 Thymol Rate Loss Thymol Rate loss (%/day) Particle CompositionFrom From Protein Carrier Fluid % Thymol 0 to 7 days 7 to 19 days 71%Gluten 14% glycerol 15 1.71 0.08 81% Gluten 14% glycerol 5 0.23 0.03 85%Gluten 14% glycerol 1 0.04 0.00

Example 14

A composition of 71% WhetPro®, 14% glycerol, 15% thymol was prepared byextrusion and downsized as described in Example 6. The particle sizerange of 250-425 micron was collected via sieving.

A series of thymol extractions were carried out from the particlesprepared in Example 14 that involved various amounts of thymol/proteinparticles, various amounts of water, addition of citric acid particles,and extracting time. The extracting method involved adding specifiedamount of water to protein/thymol particles and citric acid particles,waiting a specified amount of time while shaking, and centrifuging tocollect supernatant. In addition, water was added to neat thymol as acontrol, shaken for specified amount of time, and centrifuged to collectsupernatant. The thymol concentration in supernatant was determined bythe method described above. The composition of the solutions and theresulting thymol concentration are set forth in Table 10.

TABLE 10 Thymol Extractions from Particles Particle Composition % ThymolParticle Citric Deionized Extracting (wt/wt) Size Amount acid water timein extract Protein Carrier Fluid % Thymol (microns) (grams) (grams)(milliliters) (mins) solution 71% Gluten 14% glycerol 15% 250-425 0.750.38 12.4 30 0.557 1.5 0.38 12.4 30 1.115 0.75 0.38 12.4 10 0.509 0.75 012.4 30 0.048 2.25 1.14 37.2 30 0.610 n/a n/a 100 (control) <250 2 1.1497 30 0.081 2 0 100 30 0.084

The reported solubility limit of thymol in water is normally is 0.1grams per 100 grams of water (0.1%) at 25° C. As illustrated above,however, the concentrations of thymol in the extract solutions were upto 11 times greater than the reported limited solubility. Two of thesolutions above (0.557% thymol and 1.115% thymol) were also tested forantimicrobial efficacy according to the microplate assay describedabove. The results are set forth below in Table 11.

TABLE 11 Efficacy of Thymol Extractions # of wells # of wells Particleshowing growth showing growth Carrier % Size Amount after test for aftertest for Example Protein Fluid Thymol (microns) (grams) S. aureus P.aeruginosa 15 71% Gluten 14% glycerol 15 250-425 0.75 1 1 16 1.5 3 1

While the invention has been described in detail with respect to thespecific embodiments thereof, it will be appreciated that those skilledin the art, upon attaining an understanding of the foregoing, mayreadily conceive of variations and equivalents to these embodiments.Accordingly, the scope of the present invention should be assessed asthat of the appended claims and any equivalents thereto.

1. A method for forming an antimicrobial composition comprisingdispersively blending a botanical oil, protein, and carrier fluid withina melt blending device at a temperature of from about 20° C. to about100° C. and a shear rate of from about 1 to about 100 Pascal-seconds,wherein botanical oils constitute from about 0.1 wt. % to about 30 wt. %of the composition, proteins constitute from about 30 wt. % to about 95wt. % of the composition, and carrier fluids constitute from about 1 wt.% to about 50 wt. % of the composition.
 2. The method of claim 1,wherein the botanical oil includes a monoterpene phenol.
 3. The methodof claim 2, wherein the monoterpene phenol is thymol, carvacrol, or amixture thereof.
 4. The method of claim 1, wherein the protein is aplant protein.
 5. The method of claim 4, wherein the plant protein iswheat gluten.
 6. The method of claim 1, wherein the carrier fluid is apolyhydric alcohol.
 7. The method of claim 6, wherein the polyhydricalcohol is glycerol.
 8. The method of claim 1, wherein the carrier fluidis an aliphatic carboxylic acid.
 9. The method of claim 1, whereinbotanical oils constitute from about 0.5 wt. % to about 20 wt. % of thecomposition, proteins constitute from about 40 wt. % to about 90 wt. %of the composition, and carrier fluids constitute from about 5 wt. % toabout 30 wt. % of the composition.
 10. The method of claim 1, whereinthe composition further comprises a starch polymer.
 11. The method ofclaim 1, wherein the botanical oil, protein, and carrier fluid areblended within the melt blending device at a temperature of from about30° C. to about 80° C.
 12. The method of claim 1, wherein the botanicaloil, protein, and carrier fluid are blended within the melt blendingdevice at a shear rate of from about 5 to about 60 Pascal-seconds. 13.The method of claim 1, wherein the melt blending device is an extruder.14. The method of claim 1, further comprising extruding theantimicrobial composition onto a surface to form a film.
 15. Amelt-processed antimicrobial composition comprising at least onemonoterpene phenol in an amount of from about 0.1 wt. % to about 30 wt.%, at least one melt-processible plant protein in an amount of fromabout 30 wt. % to about 95 wt. %, and at least one carrier fluid inamount of from about 1 wt. % to about 50 wt. %.
 16. The composition ofclaim 15, wherein the monoterpene phenol is thymol, carvacrol, or amixture thereof.
 17. The composition of claim 15, wherein the plantprotein is wheat gluten.
 18. The composition of claim 15, wherein thecarrier fluid is a polyhydric alcohol.
 19. The composition of claim 18,wherein the polyhydric alcohol is glycerol.
 20. The composition of claim15, wherein the carrier fluid is an aliphatic carboxylic acid.
 21. Thecomposition of claim 15, wherein monoterpene phenols constitute fromabout 0.5 wt. % to about 20 wt. % of the composition, melt-processibleplant proteins constitute from about 40 wt. % to about 90 wt. % of thecomposition, and carrier fluids constitute from about 5 wt. % to about30 wt. % of the composition.
 22. The composition of claim 15, whereinthe composition is generally free of solvents.
 23. The composition ofclaim 15, wherein the composition is in the form of particles.
 24. Thecomposition of claim 23, wherein the particles have an average size offrom about 100 to about 600 micrometers.
 25. A wipe comprising themelt-processed antimicrobial composition of claim
 15. 26. The wipe ofclaim 25, wherein the fibrous material contains absorbent fibers. 27.The wipe of claim 26, wherein the fibrous material is a composite ofabsorbent fibers and synthetic thermoplastic fibers.
 28. A method forremoving bacteria from a surface, the method comprising contacting thesurface with a wipe that comprises a fibrous material that contains amelt-processed antimicrobial composition comprising at least onemonoterpene phenol in an amount of from about 0.1 wt. % to about 30 wt.%, at least one melt-processible plant protein in an amount of fromabout 30 wt. % to about 95 wt. %, and at least one carrier fluid inamount of from about 1 wt. % to about 50 wt. %
 29. The method of claim28, wherein the composition is in the form of particles.
 30. The methodof claim 29, wherein the particles have an average size of from about100 to about 600 micrometers.
 31. The method of claim 28, wherein priorto contacting the surface with the wipe, an aqueous solution is appliedto the composition to release the monoterpene phenol.
 32. The method ofclaim 31, wherein the concentration of the monoterpene phenol releasedin the aqueous solution is greater than about 0.1 wt. %.
 33. The methodof claim 31, wherein the concentration of the monoterpene phenolreleased in the aqueous solution is from about 1 wt. % to about 10 wt.%.
 34. The method of claim 31, wherein the aqueous solution contains anacid.